I. Organism Information

A. Taxonomy Information

1. Species:
   1. Hepatitis E virus :
      1. GenBank Taxonomy No.: 12461
      2. Description: Hepatitis E is a self-limited, enterically transmitted acute viral hepatitis that occurs most frequently in epidemic outbreaks and is etiologically associated with a newly identified and molecularly characterized virus, the hepatitis E virus (HEV). This disease, frequently spread by fecally contaminated drinking water, has also been called enterically transmitted non-A, non-B hepatitis (ET-NANB or ENANB hepatitis) (Krawczynski, 1993). The term "enterically transmitted non-A, non-B hepatitis" (ET-NANBH) derives from the waterborne mode of disease transmission and presumed enteric route of natural infection in humans (Bradley et al., 1987). The virus was discovered in 1983 by immune electron microscopy and was first cloned in 1990. It is the sole member of the genus Hepevirus in the family Hepeviridae. HEV sequences have been classified into four genotypes: genotype 1 consists of epidemic strains in developing countries in Asia and Africa; genotype 2 has been described in Mexico and Africa; genotype 3 is widely distributed in the world and has been isolated from sporadic cases of acute hepatitis E and/or domestic pigs in the USA, European countries and Japan; and genotype 4 contains strains from humans and/or domestic pigs in Asian countries including China, Taiwan and Japan (Tanaka et al., 2007). Although the sequences of different HEV isolates are heterogeneic, they all belong to a single serotype (Sun, 2005). Outbreaks of hepatitis E involving several thousand cases have been observed in the developing countries of the Indian subcontinent, Asia and Africa. Hepatitis E also accounts for a significant number of sporadic cases in endemic regions. The large number of cases involved, frequency of epidemics and high mortality rate among infected pregnant women are strong indicators that hepatitis E is an important cause of morbidity and mortality in human beings (Krawczynski, 1993).
      3. Variant(s):
         • Big liver and spleen disease virus :
           • GenBank Taxonomy No.: 301242
           • Parent: Hepatitis E virus
           • Description: Big liver and spleen disease (BLS) was recognized in Australia since 1980 and considered the most economically significant disease affecting commercial broiler breeder flocks in Australia. It causes decreased egg production and a slight increase in mortality in broiler breeder flocks. Affected birds have hepatomegaly and splenomegaly and histological lesions are characterized by a period of lymphoproliferation followed by a period of lymphoid destruction that coincides with the clinical signs (Payne et al., 1999). In 2001 a virus associated with hepatitis-splenomegaly syndrome (HS) in chickens in the USA, was partially sequenced and shown to be genetically related to hepatitis E viruses. A short region of BLS virus (BLSV) sequence was first shown to be 61% identical to human hepatitis E. The hepatitis-splenomegaly syndrome (HS) syndrome was first described in Canada and subsequently in the USA. It probably occurs worldwide and is presumably related to big liver and spleen disease (BLSD) described in Australia (Goens and Perdue, 2004). Another animal strain of HEV, designated avian HEV, has been genetically identified and characterized from chickens with hepatitis-splenomegaly (HS) syndrome in the United States. The newly discovered avian HEV is genetically related to but distinct from other known HEV strains. Unlike swine HEV, which causes only subclinical infection in pigs, avian HEV is associated with HS syndrome in chickens. HS syndrome in chickens was first described in 1991 in western Canada and is now recognized in eastern Canada and the United States (Huang et al., 2002).
         • Hepatitis E virus (isolate rhesus) :
           • GenBank Taxonomy No.: 31766
           • Parent: Hepatitis E virus
           • Description: Hepatitis E virus isolated from bile in the gallbladder of the rhesus monkey (Uchida et al., 1992).
         • Hepatitis E virus (strain Burma) :
           • GenBank Taxonomy No.: 31767
           • Parent: Hepatitis E virus
           • Description: Hepatitis E virus (strain Burma) isolated from human fecal material from a Burmese patient and cloned by Genelabs, California, USA (Reyes et al., 2002).
         • Hepatitis E virus (strain Mexico) :
           • GenBank Taxonomy No.: 31768
           • Parent: Hepatitis E virus
           • Description: HEV isolated from a human stool sample collected during an HEV outbreak in Mexico (Huang et al., 1992).
         • Hepatitis E virus (strain Myanmar) :
           • GenBank Taxonomy No.: 31769
           • Parent: Hepatitis E virus
           • Description: Hepatitis E virus strain isolated from fecal extract of patients with sporadic hepatitis in Yangon (Rangoon) in Myanmar (Burma) (Aye et al., 1993).
         • Hepatitis E virus (strain Pakistan) :
           • GenBank Taxonomy No.: 33774
           • Parent: Hepatitis E virus
           • Description: A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was isolated from the feces of a patient during a hepatitis E outbreak in Pakistan (Tsarev et al., 1992). The nucleotide sequence of the Pakistan strain of HEV is highly related to that of Burma strain except for a hypervariable region (Huang et al., 1992).
         • Hepatitis E virus type 1 :
           • GenBank Taxonomy No.: 185579
           • Parent: Hepatitis E virus
           • Description: Hepatitis E virus strain isolated feces from a female patient with hepatitis E from an epidemic of the south part of Xinjiang Uighur Autonomous Region, in which 120,000 patients occurred between September, 1986 and April, 1988 (Aye et al., 1992).
         • Hepatitis E virus type 4 :
           • GenBank Taxonomy No.: 185580
           • Parent: Hepatitis E virus
           • Description: Human isolate of HEV genotype 4. More recently, HEV genotype 4 was identified in the sera of patients, diagnosed with acute hepatitis, from various regions of China, including the south (Guangzhou and Shanghai), centre (Henan province) and north (Liaoning province and Beijing) and seems to be more common than genotype 1 as a cause of sporadic hepatitis E in China (Wang et al., 2002).

B. Lifecycle Information :

1. Virion :
   1. Size: 30-34 nm in diameter (Purcell and Emerson, 2002)
   2. Shape: Hepatitis E virus is a nonenveloped, spherical particle, approximately 30 to 34 nm in diameter, with an indefinite surface substructure that is slightly less pronounced than that of the Norwalk agent (a calicivirus) but distinguishable from the smooth, featureless surface of hepatitis A virus (a picornavirus). However, on the basis of morphology, HEV cannot be reliably distinguished from other "small round viruses" found in feces. Analysis of electron micrographs of HEV particles by Markham rotational analysis provided images that suggested an icosahedral symmetry for HEV virions (Purcell and Emerson, 2002).
   3. Picture(s):
      1. Hepatitis E virus
         
         
         
         Description: Electron microscope image of human hepatitis E virus (Centers for Disease Control, 2003) (Goens and Perdue, 2004).
         
   4. Description: Hepatitis E virus (HEV), the major etiologic agent of enterically transmitted non-A, non-B hepatitis worldwide, is a spherical, non-enveloped, single stranded RNA virus that is approximately 32 to 34 nm in diameter (CDC2006a). It is the sole member of the genus Hepevirus in the family Hepeviridae (Tanaka et al., 2007).

C. Genome Summary:

1. Genome of Hepatitis E virus
1. Description: Genomic organization of HEV: (A) Genotype 1 - 3 (B) Genotype 4 (Panda et al., 2006).
2. Chromosome:
   1. GenBank Accession Number: NC_001434
   2. Size: 7,176 nt (NCBI Entrez)
   3. Gene Count: 10 (NCBI Entrez)
   4. Description: HEV is a positive-sense, RNA virus without an envelope. The capped, single-stranded genome is approximately 7.2 kb in length and contains three open reading frames (ORFs) flanked by short non-translated regions (Zhang et al., 2006). It has three overlapping open reading frames (ORFs), with the nonstructural genes located at the 5' end of the genome and the structural genes at the 3' end. Although most of the structural proteins are coded within ORF2, all three frames contribute to the morphology of HEV (Safary, 2001). ORF 1 is the largest of the three and encodes a non-structural protein containing methyl transferase, helicase, and replicase domains (Zhang et al., 2006). It begins at the 5' end of the viral genome after 28 nucleotides of the 5' NCR, and extends for 5079 nucleotides before terminating at nucleotide position 5109 (Panda et al., 2006). The second major ORF (ORF2) that encodes the structural protein begins at nucleotide position 5147 and extends approximately 1980 nucleotides, before terminating at nucleotide position 7127 and codes for a protein of 660 amino acids (Panda et al., 2006). ORF 2 encodes a protein that contains a typical signal sequence near its 5' end, immediately followed by a region rich in codons for arginine and highly basic in charge. This region is believed to be involved in the encapsidation of the genomic transcript. The ORF 2-encoded protein contains potential glycosylation sites that are glycosylated in vitro, but it has not been established that these are functional in vivo. ORF 2 encodes a major immunogenic epitope at the extreme 3' end, as well as other important epitopes in the central region of the protein (Purcell and Emerson, 2002). A third ORF (ORF3) is 369 nucleotides in length, overlaps ORF1 at its 5' end by one nucleotide, significantly overlaps ORF2 and encodes a minor protein of 123 amino acids (Panda et al., 2006). ORF 3 encodes a phosphoprotein that contains a signal sequence near its 5' end but lacks identifiable motifs. It binds to the cellular cytoskeleton, but its function is unknown. This protein also contains an immunogenic epitope near its 3' end (Purcell and Emerson, 2002). There is a 3-nucleotide deletion in the Indian strain at nucleotide position 5302 - 5304 that results in ORF2 and ORF3 polypeptides of 659 and 122 amino acids, respectively, shorter by one amino acid than other strains. The 3' terminal 68 nucleotides comprise an untranslated region that terminates at a polyadenylated tail (Panda et al., 2006).
   5. Picture(s):
      1. Hepatitis E Virus (Panda et al., 2006):
         
         
         
         Description: HEV genome consists of a single-stranded positive sense polyadenylated RNA of approximately 7.2kb in length (Panda et al., 2006).
         

II. Epidemiology Information

Hepatitis E is a major public health concern in many developing countries, such as Asia, Africa and Latin America, and also occurs as sporadic cases in many industrialized countries including the United States (Sun, 2005). The hepatitis E virus infection was originally thought to be limited only for certain geographic areas and humans. Recently, the genetic diversity and worldwide distribution of hepatitis E virus in human population, as well as, the circulation of the virus in many species of animals has been demonstrated (Reuter and Szucs, 2004). ET-NANBH was documented in New Delhi, India, in 1955, when 29,000 cases of icteric hepatitis were identified after widespread fecal contamination of the city's drinking water. ET-NANBH was subsequently reported to occur in either epidemic or endemic forms in India, Nepal, Burma, Pakistan, the Soviet Union and northern Africa. A high mortality rate, approaching 20%, has been noted in ET-NANBH-infected pregnant women (Bradley et al., 1987). Similar cases of sporadic hepatitis, presumed to be hepatitis E, account for up to 90% of reported hepatitis in countries where hepatitis E is endemic. Furthermore, hepatitis E has been implicated in fulminant hepatitis of pregnancy (Tsarev et al., 1992). Genotype 1 HEV was responsible for a number of waterborne epidemics of hepatitis E in Asia and Africa and also is implicated in sporadic cases of hepatitis E in these continents. The closely related genotype 2 has been detected less frequently; it was responsible for two outbreaks in Mexico in 1986 and also has been implicated in sporadic infections in Nigeria. On the other hand, genotypes 3 and 4 have not been found responsible for epidemics in humans, although they do cause sporadic cases of acute hepatitis; these infections seem to be zoonotic and both genotypes have been detected in pigs (genotype 3, worldwide, and 4, in East Asia), which may constitute the major reservoir. In China, genotypes 1 and 4 are prevalent: a widespread epidemic attributable to genotype 1 occurred between 1986 and 1988 in Xinjiang and genotype 4, as well as genotype 1, cause sporadic cases (Zhang et al., 2006). Although, hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Recent evidence indicates that there exist animal reservoirs for HEV and that hepatitis E is a zoonosis. The first animal strain of HEV, swine HEV, was isolated and characterized from pigs in the United States in 1997 by Meng et al. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV (Huang, 2004). Sporadic cases and large water-borne epidemics of hepatitis E occur frequently in many subtropical developing regions of the world, such as India, China and Africa. Epidemics have not occurred in industrialized countries and, until recently, the rare sporadic cases of hepatitis E in these countries were attributed to importation following a visit to an endemic country. However, seroprevalence studies have indicated that anti-HEV is present in 1 - 20 % of the population in industrialized countries. Recent studies have demonstrated that HEV is endemic in swine worldwide (Emerson et al., 2002). HEV infection occurs in the form of epidemics of variable magnitudes as well as permanently present sporadic cases in tropical and semi-tropical countries. These countries share poor socioeconomic and hygienic conditions that facilitate the spread of HEV through contaminated water. Figure 1 provides a map of the distribution of outbreaks or confirmed HEV infection in >25% of sporadic non-ABC hepatitis. Seroprevalence studies of HEV Immunoglobulin (Ig) have shown that hepatitis E accounts for more than 50% of cases of acute viral hepatitis in young adults in developing countries. However, HEV infection is not restricted to such endemic areas, but appears to have a worldwide distribution. In industrialized countries, only rare cases of the disease have been reported, predominantly among travelers to endemic areas. Studies in nonendemic industrialized countries have shown an HEV Ig prevalence of about 2 - 3% among so-called normal human populations, a rate much higher than expected. One of the peculiar characteristics of HEV infection is that, in epidemic situations, as well as in sporadic cases, the highest incidence of infection occurs in subjects between 15 and 40 years of age with a higher incidence in males than females at a ratio of 1.5 - 3.5:1. However, in pediatric cases, males and females are equally represented (Safary, 2001). Hepatitis E virus infection manifests as epidemic and sporadic hepatitis in disease - endemic areas. Epidemics of hepatitis E occur as outbreaks involving a large number of cases. The first extensively studied outbreak of waterborne hepatitis was the Delhi outbreak in 1955 - 1956 with more than 29000 icteric cases. Since then notable epidemics have been reported. During these outbreaks, attack rates of 1 - 15% with excess cases among the 15 - 40 years of age group were seen. However, there have been other studies that have suggested a high susceptibility of children to HEV infection. The case-fatality rate during epidemics is observed to be between 0.2% and 4% but a significant high rate of 10 - 20% of unexplained fulminant liver failure has been seen in pregnant women, especially in the third trimester. In India, 30 - 60% of all sporadic cases of hepatitis are due to HEV infection. Different geographical locations worldwide, where epidemic and rampant sporadic HEV infections have been identified. The presence of locally acquired hepatitis E infections in these countries indicates HEV circulation in the environment. This has been further substantiated in Iraq (Panda et al., 2006).

A. Outbreak Locations:

1. Vietnam: A hepatitis outbreak affecting primarily adults occurred in southwestern Vietnam, along the Hau river bordering Cambodia, in June and July 1994. Immunoglobulin M to HEV was detected by enzyme-linked immunosorbent assay and Western blot in 16% of sera collected from patients one month after the outbreak. Hepatitis E virus RNA was detected with the polymerase chain reaction in 6% of sera from patients; RNA was not detected in either control group. These results indicate that HEV was the etiologic agent responsible for the outbreak. Children were under-represented among clinical cases. River water served as the principal source for drinking and bathing among most (96%) of the case and control study populations. Boiling of drinking water was negatively associated (P < 0.05) with IgG anti-HEV seropositivity. Unusually heavy rainfall likely contributed to conditions that favored the outbreak (Corwin et al., 1996).
2. Nepal: From 29 January 1995 to 15 March 1995, an outbreak of hepatitis occurred among 692 soldiers at an isolated training camp 25 km east of Kathmandu. Thirty-two cases occurred approximately 8 weeks after arrival of soldiers at the camp. To determine the etiology of the outbreak, patient sera were examined for evidence of infection with hepatitis A, B, C, and E viruses using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The polymerase chain reaction (PCR) was used to detect hepatitis E virus (HEV) RNA. Evidence of recent infection (IgM to HEV and/or HEV RNA) was found in all but two patients, whereas none had evidence of recent infection with hepatitis A, B, or C viruses. Therefore, the outbreak was attributed to HEV. Fecally contaminated drinking water was suspected as the source of the outbreak (Clayson et al., 1998).
3. Vietnam: A hepatitis outbreak affecting primarily adults occurred in southwestern Vietnam, along the Hau river bordering Cambodia, in June and July 1994. The prevalence of immunoglobulin G (IgG) to hepatitis E virus (HEV) was significantly (P < 0.001) higher (76%) among cases than among the matched (38%) and geographic (38%) control populations. Immunoglobulin M to HEV was detected in 16% of sera collected from patients one month after the outbreak. Hepatitis E virus RNA was detected in 6% of sera from patients; RNA was not detected in either control group. These results indicate that HEV was the etiologic agent responsible for the outbreak. Children were under-represented among clinical cases. River water served as the principal source for drinking and bathing among most (96%) of the case and control study populations. Boiling of drinking water was negatively associated (P < 0.05) with IgG anti-HEV seropositivity (Corwin et al., 1996).
4. Sudan: In June 2004, a large hepatitis E outbreak occurred in western Darfur, Sudan (Boccia et al., 2006). In 6 months, 2621 hepatitis E cases were recorded, with a case-fatality rate of 1.7% (45 deaths, 19 of which involved were pregnant women). Risk factors for clinical HEV infection included age of 15 - 45 years and drinking chlorinated surface water (Guthmann et al., 2006).
5. Mexico: Outbreaks of acute hepatitis occurred in Huitzililla and Telixtac, two rural villages 70 miles south of Mexico City, Mexico, in late 1986. The first outbreak began in Huitzililla in June of that year, 1 month after the start of the rainy season in which two women died. The second outbreak began in August 1986 in Telixtac in which one woman died. Two of eight stool samples from Huitzililla and one of the eight stool samples from Telixtac were positive by immune electron microscopy for 32- to 34-nm virus-like particles similar to those seen in cases of enterically transmitted non-A, non-B hepatitis from Asia (Velazquez et al., 1990).
6. Pakistan: An epidemic of enterically transmitted non-A, non-B hepatitis occurred at a college in Sargodha, Pakistan in early 1987. There were 133 clinical cases, an attack rate of approximately 20%. Though the disease was relatively mild, all clinical cases required hospitalization and prolonged convalescence. Nearly all cases were associated with a single water source. The epidemic ended when the water supply was improved (Iqbal et al., 1989).
7. Nigeria: Sporadic cases of acute hepatitis E among ten native Nigerian adults were reported in Port-Harcourt (Nigeria). Diagnosis of the ten sporadic cases of non-A, non-B acute hepatitis was suggested by the self-limited course of the disease. Serological evidence of HEV came from the strong anti-HEV reactivity with anti-HEV IgM antibodies in all ten patients. Hepatitis E virus (HEV) was detected in serum and/or fecal samples of seven patients by RT-PCR of the open reading frame (ORF)-1 polymerase region and the 3'-end of ORF2 (Buisson et al., 2000).
8. Myanmar: An epidemic outbreak of hepatitis E occurred in an army recruit camp of Yangon, Myanmar, in October 1989. One hundred and eleven patients among 600 residents were hospitalized. As high as 83.7% of these patients were positive for the acute phase antibody against hepatitis E virus by an enzyme-linked immunosorbent assay (Uchida et al., 1993).

B. Transmission Information:

1. From: Animals To: Human
   Mechanism: Sporadic cases of HEV constitute a potential reservoir for maintaining the virus in the population, and extended periods of virus excretion may occur in some of those infected. Moreover, other mammalian hosts may constitute important reservoirs of the virus. Recent data suggest that hepatitis E may be a zoonosis. HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals. In addition, human HEV isolates have been shown to be phylogenetically related to swine HEV from US, Taiwan, New Zealand and Japan and possess cross-species infectivity. Definitive evidence for animal to human transmission has come from disease in individuals consuming deer meet and isolation of identical virus from remnant frozen meat (Panda et al., 2006). Recently, HEV strains have been isolated from swine in industrialized countries. In addition, cases of acute hepatitis due to novel HEV variants have been reported in humans without recognized risk factors for hepatitis E in the US, Japan and Europe. Some of the novel strains were found to be closely related to swine HEV isolates from the same area, suggesting that hepatitis E is a zoonotic disease. Thus hepatitis E is becoming an issue in countries where HEV is not, traditionally, believed to be endemic (Worm et al., 2002).
   
   
2. From: Water To: Human
   Mechanism: The primary mode of transmission of HEV is faecooral. Evidence of fecal contamination of drinking water supplies has been associated with several HEV epidemics in India; during winter months when the water level falls, concentrating the contaminants, and during the monsoon season when there is flooding and increased chances of fecal contamination of drinking water. The detection of HEV genomic sequence in raw and treated sewage water and in the stools of patients during HEV epidemics further substantiates the enteric mode of HEV transmission (Panda et al., 2006).
   
   
3. From: Food To: Human
   Mechanism: Recent studies have indicated that zoonotic food-borne transmission of HEV from domestic pigs, wild boar or wild deer to humans may occur as domestic infection in Japan, where some people ingest uncooked or undercooked meat or viscera (such as raw liver and colon/intestines). Of interest, one swine HEV isolate (swJL145) obtained from a packaged pig liver was 100 % identical to the virus recovered from an 86-year-old patient who had contracted sporadic hepatitis E after ingestion of undercooked pig liver, suggesting that consumption of undercooked pig liver/intestine is a potential risk factor for HEV infection (Tanaka et al., 2007). More recently, members of a family group were infected following consumption of uncooked Sika deer meat. The virus identified from the patients was shown to be identical to that recovered from uneaten quantities of meat from the same deer (Banks et al., 2004). Development of acute hepatitis E by a Japanese patient after ingestion of Chinese herbal medicine has also been documented (Panda et al., 2006).
   
   
4. From: Human To: Human
   Mechanism: HEV is transmitted rarely through person-to-person transmission. The first documented hepatitis E outbreak occurred in Delhi, India, in 1955 - 1956. Additional outbreaks have been reported among civilians and military populations (Boccia et al., 2006). It is evident, that hepatitis E virus can spread even by transfusion (blood-borne transmission). The frequency of hepatitis E virus infections among the acute hepatitis cases with unknown origin is supposed to be more than 10% (Reuter and Szucs, 2004). Limited information is available on alternative routes of HEV transmission. Intra-familial or contact transmission of HEV appears to be of minor significance. Secondary attack rates among household members of patients with acute hepatitis E occur in about 1 - 2% of the cases. Protracted viremia has been reported in a small group of patients suffering from acute hepatitis E, though the viraemic phase of HEV infection is usually brief. Recently, prolonged viremia for more than three years has been detected in patients with renal transplants and immunosupportive therapy (Panda et al., 2006). Two recent studies, one in Japan and the other in Saudi Arabia, suggest that HEV may also be transmitted parenterally (Goens and Perdue, 2004). The probability of parenteral transmission is possibly low and reports have been restricted to nosocomial spread including a hospital outbreak, in which the disease was transmitted to the medical staff attending an acute hepatitis E patient. Moreover, IgG anti-HEV, a post-infection marker of Hepatitis E, is frequently detected in-groups at risk for parenteral transmission e.g. transfusion recipients haemodialysis patients, intravenous drug abusers. Vertical transmission of HEV from mother to child during the third trimester of pregnancy and in 50% of HEV infected pregnant women has been reported. There is no evidence for sexual transmission of HEV, although in a report from Italy, 20% homosexual men had anti-HEV antibodies as compared to only 3% of intravenous drug users (Panda et al., 2006).
   
   

C. Environmental Reservoir:

1. Rodent :
   1. Description: Murid rodents and house shrews trapped in Nepal's Kathmandu Valley, where hepatitis E is hyperendemic, were tested for HEV infection. The most commonly trapped species was Rattus rattus brunneusculus. Serum samples from 675 animals were tested for immunoglobulin G against HEV by enzyme-linked immunosorbent assay; 78 (12%) were positive, indicating acute or past infection. Antibody prevalence was higher among R. rattus brunneusculus and Bandicota bengalensis than in Suncus murinus. Forty-four specimens from 78 antibody-positive animals had sufficient residual volume for detection of HEV RNA (viremia) by reverse transcription-PCR. PCR amplification detected four animals (9%; three were R. rattus brunneusculus and one was B. bengalensis) with viremia. Phylogenetic analysis of the four genome sequences (405 bp in the capsid gene) recovered showed that they were identical, most closely related to two human isolates from Nepal (95 and 96% nucleotide homology, respectively), and distinct from HEV sequences isolated elsewhere. These data prove that certain peridomestic rodents acquire HEV in the wild and suggest that cross species transmission occurs, with rodents serving as a virus reservoir for humans (He et al., 2002).
2. Wild boar :
   1. Description: In Japan, wild boars and deer are infected with HEV, although at much lower rates than domestic pigs. In addition, certain proportions of wild boars in Japan have HEV and may act as sources of HEV infection in humans. The isolation of a domestic strain of HEV from a Japanese wild boar with high nucleotide sequence identity to human HEV in Japan provides further evidence for zoonotic food-borne transmission of HEV from wild boars to humans (Sonodaet al., 2004). HEV infection is endemic in wild boar in the Ehime area, and should be regarded as an important reservoir of HEV (author et al., 2007).
3. Outbreaks :
   1. Description: Sporadic cases of HEV constitute a potential reservoir for maintaining the virus in the population, and extended periods of virus excretion may occur in some of those infected (Panda et al., 2006).
4. Domestic swine :
   1. Description: Genotypes 3 and 4 of HEV have not been found responsible for epidemics in humans, although they do cause sporadic cases of acute hepatitis; these infections seem to be zoonotic and both genotypes have been detected in pigs (genotype 3, worldwide, and 4, in East Asia), which may constitute the major reservoir (Zhang et al., 2006). In addition, human HEV isolates have been shown to be phylogenetically related to swine HEV from US, Taiwan, New Zealand and Japan and possess crosss-pecies infectivity (Panda et al., 2006).
5. Cattle :
   1. Description: Mammalian hosts may constitute important reservoirs of the virus. Recent data suggest that hepatitis E may be a zoonosis. HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
6. Donkey :
   1. Description: Recent data suggest that hepatitis E may be a zoonosis. HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
7. Deer :
   1. Description: Other mammalian hosts may constitute important reservoirs of the virus. Recent data suggest that hepatitis E may be a zoonosis. HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals. Definitive evidence for animal to human transmission has come from disease in individuals consuming deer meet and isolation of identical virus from remnant frozen meat (Panda et al., 2006).
8. Mule :
   1. Description: Other mammalian hosts may constitute important reservoirs of the virus. Recent data suggest that hepatitis E may be a zoonosis. HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
9. Mongoose :
   1. Description: HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).

D. Intentional Releases:

1. Intentional Release information :
   1. Description:
   2. Emergency contact: Hepatitis is a reportable disease in all states. The goals of hepatitis surveillance at the national, state, and local levels include a) identifying contacts of case-patients who might require post-exposure prophylaxis; b) detecting outbreaks; c) determining the effectiveness of hepatitis vaccination; d) monitoring disease incidence by identifying acute, symptomatic infections in all age groups; e) determining the epidemiologic characteristics of infected persons, including the source of infection; and f) determining missed opportunities for vaccination. Cases of hepatitis should be reported to local or state health departments (according to specific state requirements) so that appropriate control measures can be implemented, if indicated. Cases meeting specified criteria are reported by state health departments to CDC. Hepatitis surveillance must be maintained at the local level so that the various recommended immunization strategies can be implemented and their outcome at the local, state, and national levels can be assessed. Laws requiring laboratories to promptly report all IgM anti-HAV positive results are likely to improve the completeness and timeliness of reporting (CDC, 1999).
   3. Delivery mechanism:
   4. Containment: Biosafety level 2 practices and containment for activities with infected materials; Animal Biosafety level 2 for activities using naturally or experimentally infected chimpanzees. Laboratory coat; gloves when direct contact with infectious materials is unavoidable; gloves and gown for work in biosafety cabinet. Animal care personnel should wear gloves and take other appropriate precautions to avoid possible fecal-oral exposure; good personal hygiene and thorough washing of hands (MSDS, Public Health Agency of Canada, 2001).

III. Infected Hosts

1. Human:
   1. Taxonomy Information:
      1. Species:
         1. Human; man :
            • GenBank Taxonomy No.: 9606
            • Scientific Name: Homo sapien (NCBI Taxonomy)
            • Description: Meng and co-workers found that swine veterinarians from eight U.S. states from which normal blood donors were also available had an increased risk of HEV infection compared to the normal U.S. blood donors. In Taiwan, where HEV was not considered to be endemic, Hsieh et al. found that about 27% of Taiwanese pig handlers were positive for anti-HEV, compared to only about 8% of control subjects. Taken together, these seroepidemiological data suggest that swine veterinarians and other pig handlers may be at potential risk of zoonotic HEV infection. The source of the relatively high anti-HEV prevalence in normal U.S. blood donors is not known. Previously, Thomas et al. reported that IgG anti-HEV was detected in about 21% of normal blood donors from Baltimore, Md. Our data confirm that such high rates occur and, in fact, are common in many states. In Japan, another country where HEV is not endemic, the prevalence of IgG anti-HEV in healthy individuals was found to range from 1.9 to 14.1%, depending on the geographic location (Meng et al., 2002).
      
      
   2. Infection Process:
      1. Infectious Dose: The infective dose is not known (Panda et al., 2006).
      2. Description: Clinical manifestations of HEV infection are similar to those of infection with other hepatitis viruses and encompass a wide spectrum of symptoms. The infection may be entirely asymptomatic, or may resemble an acute viral febrile illness without any characteristic features. When symptomatic, it typically causes acute self-limiting viral hepatitis, which runs a course of a few weeks. In some patients, hepatitis may run a prolonged cholestatic course. The pre-icteric phase lasts for 1 - 10 days, (average 3 - 4 days) and gastrointestinal symptoms such as epigastric pain, nausea, and vomiting are frequently reported. The icteric phase begins abruptly, with the appearance of jaundice, dark urine and clay colored stools. In uncomplicated cases, this lasts 12 - 15 days, and complete recovery usually takes place within 1 month (Panda et al., 2006).
      3. • Geographic Distribution of Hepatitis E (Outbreaks or Confirmed Infection in >25% of Sporadic Non-ABC Hepatitis)
           
           
           
           Description: Outbreaks of hepatitis E have occurred over a wide geographic area, primarily in developing countries with inadequate environmental sanitation. The reservoir of HEV in these areas is unknown. The occurrence of sporadic HEV infections in humans may maintain transmission during inter-epidemic periods, but a nonhuman reservoir for HEV is also possible. In the United States and other non-endemic areas, where outbreaks of hepatitis E have not been documented to occur, a low prevalence of anti-HEV (<2%) has been found in healthy populations. The source of infection for these persons is unknown. (Note: The map of HEV infection generalizes available data and patterns may vary within countries.) (NCBI Taxonomy)
           
         • Hepatitis E Virus Infection
           
           
           
           Description: The typical serologic course following HEV infection has been characterized using experimental models of infection in nonhuman primates and human volunteer studies. In two human volunteer studies, liver enzyme elevations occurred 4 - 5 weeks after oral ingestion and persisted for 20 - 90 days. Virus excretion in stools occurred approximately 4 weeks after oral ingestion and persisted for about 2 weeks. Both IgM and IgG antibody to HEV (anti-HEV) are elicited following HEV infection. The titer of IgM anti-HEV declines rapidly during early convalescence; IgG anti-HEV persists and appears to provide at least short-term protection against disease (CDC2006b).
           
      
      
   3. Disease Information:
      1. Hepatitis E (i.e., Acute hepatitis; enterically transmitted non-A non-B hepatitis (ET-NANBH). Other names include fecal-oral non-A non-B hepatitis, and A-like non-A non-B hepatitis) :
         1. Pathogenesis Mechanism: Although the hepatitis viruses cause liver damage, none is directly cytopathic to hepatocytes (liver cells). Following acute liver injury, the clinical manifestations and outcome of viral hepatitis are actually determined by the host immune response. Because serological (antibody-based) assays for HEV have only recently become available, the pathogenesis of hepatitis E is not well understood. Following the entry of HEV into the host via the oral route, the primary site of replication is probably in the intestinal tract. It is still not clear how the virus reaches the liver, but it is presumably via the portal vein serving the liver. HEV replicates in the cytoplasm of hepatocytes and is released into the bile and bloodstream by mechanisms that are not understood (Jameel, 1999). Based upon limited studies of oral infection in a human volunteers, viremia was first detected on day 22 post-exposure, about a week prior to the onset of icterus, which was around day 30. Virus-like particles were detected in the feces by day 34, post-exposure. Anti-HEV antibodies in a volunteer were first detected on day 41 and were detectable even 2 years later (Panda et al., 2006). The most salient feature of HEV infections is the increased severity of disease in pregnant women. While there does not appear to be an increased incidence of HEV infection in pregnant women, there is a clear increase in the incidence of fulminant hepatic failure (FHF) complicated by encephalopathy and disseminated intravascular coagulation in HEV-infected pregnant women. This increase in disease severity is reflected by an increase in mortality rate to over 20% in pregnant women (0.5 - 4% in men and non-pregnant women). This mortality rate also increases with gestation, as the incidence of FHF almost doubles after the first trimester. In two independent studies 62% and 64% of HEV-infected pregnant women developed FHF. While there is a 53% mortality rate in women with FHF during pregnancy in the absence of HEV infection, the mortality rate with HEV-associated FHF in the second study was 100% (Goens and Perdue, 2004).
         
         
         
         2. Incubation Period: The incubation period ranges from 15 to 60 days with a mean of 40 days. In the case of a human volunteer, clinical symptoms developed 36 days after oral intake of infected fecal samples (Panda et al., 2006). The average incubation period is 6 wk, with a range of 2 to 9 wk (Krawczynski, 1993). The incubation period to peak serum levels of liver enzymes is generally 3 to 8 weeks, but shorter and longer incubation periods have been observed. Enzyme elevations are usually unimodal, but bimodal curves have also been observed. Peak viremia and peak shedding of HEV into the feces occurs during the incubation period and early acute phase of disease (Purcell and Emerson, 2002).
         
         
         
         3. Prognosis: Hepatitis E is a self-limiting, acute disease that is clinically indistinguishable from that caused by hepatitis A virus. Overall, mortality is less than 1 %, with the exception of cases in pregnant women, which can reach a mortality of 20 % (Emerson et al., 2002). During HEV epidemics, an unusual high rate of fulminant hepatitis and subsequent mortality has been observed amongst pregnant women. Fulminant hepatic failure (FHF) develops because of encephalopathy within 8 weeks (in some cases 4 weeks) of the onset of symptoms of liver disease. HEV is also responsible for sporadic FHF in adults and children. HEV is associated with non-A, non-B liver failure, particularly in India. Travelers to areas of endemicity are at risk of acquiring infection, which may manifest as FHF when they return to their home country. Reports describing chronicity due to HEV infection have not been documented so far apart from individual cases with immunodeficiency post-kidney transplant. However, persistence of IgM anti-HEV for at least 21 months in one case and protracted viremia (45 - 112 days) with fecal shedding up to the seventh week of illness have been reported in at least four patients. HEV infection is a frequent cause of liver decompensation in patients with cirrhosis in HEV-endemic regions. Superinfection with HEV in patients with chronic liver disease causes severe liver decompensation, which is frequently complicated by hepatic encephalopathy and renal failure. Acute hepatitis E in these patients has a protracted course with high morbidity and mortality (Panda et al., 2006).
         
         
         
         4. Diagnosis Overview: HEV is responsible for the majority of ET-NANBH in endemic areas. Therefore, laboratory testing for HEV should be the first-line of diagnostic work-up in such areas for patients of hepatitis. In areas with a very low incidence of acute hepatitis E, such as Europe and the US, a patient with hepatitis and history of recent travel to an endemic area should be assessed for HEV infection. Patients without a recent travel history can be tested after ruling out more common causes of hepatitis and cholestasis. Several methods have been available for the diagnosis of hepatitis E. These include Immune electron microscopy (IEM), fluorescent antibody blocking assay, PCR, EIA and a recently developed immunochromatographic assay. Amongst these methods, EIA and immunochromatography are most convenient for the detection of IgM and/or IgG anti-HEV. These are also inexpensive and suitable assays for routine diagnosis and seroepidemiological surveys. Testing is recommended in early acute-phase of the disease to avoid false negative results. A positive result for anti-HEV IgM indicates acute HEV infection. The presence of high or increasing titre of anti-HEV IgG may additionally support the diagnosis of acute HEV infection and in such cases acute hepatitis E can be presumed even in the absence of IgM anti-HEV. However, the role of these tests in accurate diagnosis of HEV infection in endemic areas needs further evaluation. Additional testing by RT-PCR has a limited confirmatory role. In most instances, there is a very good co-relation between the positivity in RT-PCR and EIA. However, there are several limitations of EIA and the sensitivity of such assays in an endemic area for disease diagnosis remains undetermined, particularly during epidemics (Panda et al., 2006). The detection of HEV RNA in serum or fecal samples using RT-PCR has replaced the detection of virus in fecal specimens by immunoelectron microscopy (IEM) as the "golden standard" for diagnosis of HEV infection because IEM is inconvenient and its sensitivity is insufficient for routine analysis. However, because sample treatment for RT-PCR is time-consuming and expensive, and the PCR system is easily contaminated and difficult to control, the assay is available only in specialist laboratories. The routine diagnosis of hepatitis E therefore is dependent on the detection of antibodies to the virus using enzyme immunoassays (EIA), particularly for HEV-specific immunoglobulin (Ig) G and M and, to a lesser extent, IgA. The levels of anti-HEV IgG may be very high in the acute phase and decline rapidly during the convalescent period, although the antibody may remain detectable for many years. Therefore, the acute phase of HEV infection may be recognized only by increasing titers of anti-HEV IgG if that antibody is used as the sole marker for the diagnosis of acute HEV infection. Anti-HEV IgM and IgA can be detected in more than 95% and 50% of sera in the acute phase, respectively, and disappear early in the convalescent period, therefore their detection may be useful to differentiate the acute phase of HEV infection. However, there may be a window period of HEV replication prior to the appearance of specific antibodies. An EIA capable of detecting HEV antigen would be a valuable alternative to RT-PCR for the direct detection of the virus and early diagnosis of infection (Zhang et al., 2006).
         
         
         
         5. Symptom Information :
            • Syndrome -- Acute hepatitis:
              • Description: Infection with HEV closely resembles that with the hepatitis A virus, both in its pattern of transmission and because it usually causes an acute, self-limiting illness without chronic sequelae. Jaundice is usually accompanied by malaise, anorexia, abdominal discomfort and hepatomegaly. Clinical signs and symptoms of the acute, icteric phase, which, in humans, lasts about 14 days, are usually preceded by a prodromal phase of about 1 week characterized by fever and nausea. Hyperbilirubinemia and elevated aminotransferase levels generally resolve within 3 weeks (range 1 - 6 weeks) after onset of illness. In certain cases, HEV presents as a severe rapidly advancing disease, which often ends in death due to acute hepatic failure. Overall, the severity of illness increases with age and the overall case-fatality rate ranges from 1 to 3%. A singular feature of hepatitis E is the high mortality rate among infected pregnant women (20 - 30%), primarily those in their third trimester. In pregnant women, the increasing liver dysfunction frequently leads to abortion or premature delivery, which in turn aggravates the disease. Cases of asymptomatic HEV infection are not well defined. However, subclinical forms of the disease are suspected to be common as indicated by serologic documentation of antibody to HEV (anti-HEV) in humans who had no history of an icteric illness and the frequent occurrence of elevated aminotransferase levels in Indian household contacts around the diagnosed hepatitis E case (Safary, 2001).
              
              
              Symptoms Shown in the Syndrome:
              
              
              • Jaundice (Purcell and Emerson, 2002):
                • Description: Hepatitis E has symptoms of a self-limited, acute, icteric disease similar to those of hepatitis A. Jaundice is usually accompanied by malaise, anorexia, abdominal discomfort and liver enlargement (Krawczynski, 1993).
              • Anorexia (Purcell and Emerson, 2002):
              • Hepatomegaly (Purcell and Emerson, 2002):
              • Abdominal pain and tenderness (Purcell and Emerson, 2002):
              • Nausea (Purcell and Emerson, 2002):
              • Vomiting (Purcell and Emerson, 2002):
              • Fever (Purcell and Emerson, 2002):
                • Description: About half the patients develop fever and two-thirds complain of arthralgia (Panda et al., 2006).
            • Scleral icterus (Corwin et al., 1996):
              • Description: The principal signs and symptoms from among a 50 case subjects in southwestern Vietnam were scleral icterus (94%), dark urine (92%), and jaundice (92%) (Corwin et al., 1996).
            • Dark urine (Corwin et al., 1996):
            • Arthralgia:
              • Description: About two-thirds of patients complain of arthralgia (Panda et al., 2006).
         
         
         6. Treatment Information:
            • Management of fulminant hepatitis E: There is no specific treatment of hepatitis E. Aggressive management of fulminant hepatitis E may reduce the mortality of this complication in pregnant women (Purcell and Emerson, 2002). In areas with endemic hepatitis E virus (HEV), acute liver failure secondary to hepatitis E infection is common in pregnancy and associated with a mortality rate of up to 20%. However, there is little information on the clinical course of severe hepatitis E infection during pregnancy in non-endemic areas. Hussaini and co-workers reported two cases of severe hepatitis E in pregnancy in patients returning from the Indian subcontinent. The first case describes acute hepatic failure, with coagulopathy and encephalopathy, warranting intensive therapy and elective ventilation. In the other case, the patient had severe hepatitis with coagulopathy. Both cases spontaneously resolved with no fetal loss, highlighting the need for suspicion of HEV infection in patients returning from endemic areas and presenting with acute non-A non-B hepatitis, especially when pregnant. Furthermore, the intensive treatment of acute liver failure caused by HEV may reduce the high mortality reported in Asia (Hussaini et al., 1997).
      
      
   4. Prevention:
      1. Vaccination:
         • Description: In a high-risk population, the rHEV vaccine was effective in the prevention of hepatitis E (Shrestha et al., 2007).
         • Efficacy:
           • Rate: The safety and efficacy of an HEV recombinant protein (rHEV) vaccine in a phase 2, randomized, double-blind, placebo-controlled trial was evaluated in Nepal. 2000 healthy adults susceptible to HEV infection who were randomly assigned to receive three doses of either the rHEV vaccine or placebo at months 0, 1, and 6. The primary end point was the development of hepatitis E after three vaccine doses. A total of 1794 subjects (898 in the vaccine group and 896 in the placebo group) received three vaccine doses; the total vaccinated cohort was followed for a median of 804 days. After three vaccine doses, hepatitis E developed in 69 subjects, of whom 66 were in the placebo group. The vaccine efficacy was 95.5% (95% confidence interval [CI], 85.6 to 98.6). In an intention-to-treat analysis that included all 87 subjects in whom hepatitis E developed after the first vaccine dose, 9 subjects were in the vaccine group, with a vaccine efficacy of 88.5% (95% CI, 77.1 to 94.2) (Shrestha et al., 2007).
           • Duration:
      2. Interdiction:
         • Description: Improved personal hygiene has resulted in a marked reduction of hepatitis A virus infections. It is assumed that such changes have had a similar, and probably earlier, effect on the incidence of hepatitis E. Indeed, hepatitis occurring in Europe prior to the 20th century, and believed then to have been hepatitis A, had the epidemiologic characteristics of hepatitis E. Thus, HEV, reportedly more labile and shed in lower titers than HAV, may have disappeared from more industrialized countries in the recent past, just as HAV is currently diminishing in importance in these countries. However, regions where HAV is still highly endemic may also harbor endemic HEV (Purcell and Emerson, 2002).
         • Efficacy:
           • Rate:
           • Duration: The high prevalence of anti-HEV in domestic and wild animals, even in industrialized countries, suggests that possible zoonotic spread of HEV may continue in such regions, despite improved sanitation (Purcell and Emerson, 2002).
      
      
   5. Model System:
      1. Tamarins/ Cynomolgus macaques:
         1. Model Host: Tamarins
         2. Model Pathogens:
            • Hepatitis E virus .
         3. Description: An experimental model of enterically transmitted non-A, non-B hepatitis (ET-NANBH) was established in tamarins (Saguinus mystax mystax) and cynomolgus macaques (Macaca fascicularis). First-passage animals were inoculated with two different stool suspensions obtained from human patients with well-defined ET-NANBH. Both inocula contained 27- to 34-nm-diameter virus-like particles (VLPs) that were specifically aggregated by acute-phase ET-NANBH sera. ET-NANBH was subpassaged in both tamarins and cynomolgus macaques by using pools of stool suspensions from first-passage animals. One additional passage of disease in cynomolgus macaques resulted in a significantly shortened incubation period and increased severity of disease. VLPs similar to those found in the human inocula were observed in stool specimens of first-, second-, and third-passage cynomolgus macaques and in first- and second-passage tamarins. Our findings indicate that cynomolgus macaques are particularly suitable experimental models for studies of human ET-NANBH (Bradley et al., 1987).
      2. Cynomolgus monkey/ Owl monkey:
         1. Model Host: Cynomolgus monkeys Owl monkeys
         2. Model Pathogens:
            • Hepatitis E virus .
         3. Description: Owl and cynomolgus monkeys were inoculated with hepatitis E virus (HEV) to compare disease models and produce antibody and virus. By immune electron microscopy (IEM), all six owl monkeys were shown to have serologic responses manifested by unusually high levels of anti-HEV at 6 months, but only three developed hepatitis. Virus-related antigen in liver (HEV Ag) was detected by immunofluorescence microscopy of biopsies from two of four owl monkeys; one with HEV Ag also had HEV in acute-phase bile (detected by IEM) and feces (detected by infecting another owl monkey). In contrast, cynomolgus monkeys propagated HEV to higher levels and all five had hepatitis. Moderate-to-high levels of HEV Ag correlated with detectable HEV in bile for both species. Owl monkeys were shown to be HEV-susceptible and sources of high-level anti-HEV (Ticehurst et al., 1992).
      3. Cynomolgus macaques:
         1. Model Host: Cynomolgus macaques
         2. Model Pathogens:
            • Hepatitis E virus .
         3. Description: Cynomolgus macaques were inoculated intravenously with bile or feces containing hepatitis E virus (HEV). Serum, bile, and liver specimens were evaluated with light microscopy, immune electron microscopy, immunofluorescence microscopy, EIA, and polymerase chain reaction. In the third week, there were histopathologic changes and HEV antigen (HEVAg) in liver, HEV in bile, and alanine aminotransferase (ALT) elevations. Widespread pathologic changes were detected during the fourth week and antibody to HEV (anti-HEV) and peak ALT values in the fifth or sixth week. By the sixth week, HEVAg had disappeared but pathologic changes persisted (Longer et al., 1993). An HEV-containing fecal sample from Chad was intravenously inoculated in four cynomolgus macaques. When serum Alanine Amino Transferase (ALT) levels rose, open liver biopsy and bile aspiration were performed. In all the monkeys, an ALT rise occurred 25 to 32 days after inoculation and new anti-HEV was detected by Enzyme Immuno Assay (EIA). Hepatic histopathology was consistent with acute viral hepatitis. HEV was detected by polymerase chain reaction (PCR) in bile (3/4 animals) and feces (2/4 animals) and by imunoelectron microscopy (IEM) in the inoculum and one bile specimen. A genetic variant HEV was identified in one monkey. The Chad HEV produced hepatitis E with pathophysiologic and histopathologic findings similar to those observed with HEV from other geographic origins (van Cuyck-Gandre et al., 1998). The hypothesis that serial subclinical transmission among susceptible humans may serve as a reservoir of hepatitis E virus (HEV) in areas in which HEV is endemic was investigated in an experimental primate model. Four groups of 4 cynomolgus macaques each were inoculated intravenously with 10(4)-10(5) (group 1), 10-100 (group 2), and 1-10 (group 3) cynomolgus macaque HEV infectious doses. All 4 animals in group 1 had clinical disease marked by alanine aminotransferase (ALT) elevation, fecal virus excretion, viremia, and seroconversion. Of the animals in groups 2 and 3, only 1 had evidence of biochemical hepatitis, although most had virus excretion and viremia (3 animals each in groups 2 and 3), and evidence of seroconversion (1 animal in group 2 and 3 animals in group 3). Viral genomic titers in stool specimens of animals with or without ALT elevation were similar. Infectivity studies confirmed the viability and transmission potential of the virus excreted by animals without ALT elevation. These data suggest that subclinical HEV infection may represent an HEV reservoir (Aggarwal et al., 2001).
      4. Rhesus monkey:
         1. Model Host: Rhesus monkeys
         2. Model Pathogens:
            • Hepatitis E virus .
         3. Description: Hepatitis E virus (HEV) particles present in the stool of an acutely ill patient was transmitted intravenously into rhesus monkeys (M. mulata) and orally to a human volunteer. Virus-like particles (VLPs) of 32-34 nm were detected in the bile of monkeys and in the stools of the human volunteer by means of solid phase immune electron microscopy (SPIEM) with acute homologous and heterologous sera. The VLPs were confirmed to be HEV by a reverse transcription polymerase chain reaction (RT-PCR). Virus-like particles from human volunteer stools were passaged further into rhesus monkeys. A bimodal rise in aminotransferase levels were observed in the animals, and liver histopathology indicated mild to severe form of hepatitis. Further, SPIEM and RT-PCR analysis in monkey bile revealed presence of virus from 15 to 45 days post-inoculation. Rechallenge of the animals 6 months after recovery with the same viral inoculum failed to produce abnormal liver function tests indicating the presence of protective immunity during this period (Chauhan et al., 1994). To determine if rhesus monkeys infected with one isolate of hepatitis E virus (HEV) were immune to subsequent challenge with other isolates of the virus. Three epidemic and one sporadic Indian HEV isolates were employed in the study. The interval between primary inoculation and challenge varied from 1 year and 6 months to 2 years and 9 months. Evidence of HEV infection was ascertained by rise in serum alanine transaminase (ALT) levels and/or seroconversion to antibodies to HEV (anti-HEV), and the presence of HEV-RNA in the bile or feces of the infected monkeys. No evidence for multiplication of virus in monkeys challenged with different HEV isolates was obtained. These results show that immunity generated by one isolate of HEV protects against different isolates of hepatitis E virus (Arankalle et al., 1995).
      5. Chimpanzee:
         1. Model Host: Chimpanzees
         2. Model Pathogens:
            • Hepatitis E virus .
         3. Description: Different patterns of disease were observed among 11 chimpanzees who were inoculated intravenously with hepatitis E virus (HEV) positive fecal specimens from four different outbreaks. Five chimpanzees had marginal or no liver enzyme elevations within 70 days of inoculation. Two of the chimpanzees had limited viremia, but did not produce detectable antibody. The four remaining chimpanzees had liver enzyme elevations, viral shedding, viremia, seroconversion to anti-HEV, and detectable HEV antigen in liver biopsy specimens. These results may reflect the range of infection patterns that develop in humans after natural exposure to the HEV (McCaustland et al., 2000).
2. Animals:
   1. Taxonomy Information:
      1. Species:
         1. Metazoans; animalia; multicellular animals; animals :
            • GenBank Taxonomy No.: 33208
            • Scientific Name: metazoans (NCBI Taxonomy)
            • Description: The source of HEV infection in industrialized countries is not known, but increasing evidence supports the hypothesis of a zoonotic infection. Many different serological tests have detected anti-HEV in animals (Meng et al., 2002).
            • Variant(s):
              • Sus scrofa :
                • GenBank Taxonomy No.: 9823
                • Scientific Name: Sus scrofa (NCBI Taxonomy)
                • Common Name: Pig; wild boar; swine; pigs (NCBI Taxonomy)
                • Description: Anti-HEV has been detected in about 33% of domestic swine in Nepal, where hepatitis E is endemic. In the United States, anti-HEV has been detected in more than 80% of pigs older than 3 months of age but in very few animals younger than that age (Meng et al., 2002). The discovery of a novel virus in pigs, designated the 'swine hepatitis E virus', has been reported in the United States. These findings indicate that HEV is a zoonotic virus and that these animals are among it natural hosts which constitutes a potential public health concern (Safary, 2001). In addition, human HEV isolates have been shown to be phylogenetically related to swine HEV from US, Taiwan, New Zealand and Japan and possess cross-species infectivity (Panda et al., 2006).
              • Bos taurus; Bos primigenius taurus; Bos bovis :
                • GenBank Taxonomy No.: 9913
                • Scientific Name: Bos taurus; Bos primigenius taurus; Bos bovis (NCBI Taxonomy)
                • Common Name: Cattle; cow; bovine; domestic cattle; domestic cow (NCBI Taxonomy)
                • Description: Recently, Favorov et al. found anti-HEV in about 29 to 62% of cows from three countries where HEV is endemic (Somalia, Tajikistan, and Turkmenistan) and in 12% of cows in a country where HEV is not endemic(Ukraine) (Meng et al., 2002).
              • Capra hircus; Capra aegagrus hircus :
                • GenBank Taxonomy No.: 9925
                • Scientific Name: Capra hircus; Capra aegagrus hircus (NCBI Taxonomy)
                • Common Name: Goat; goats; domestic goat; African dwarf goats; Naine d'Afrique de l'Ouest; African dwarf goat (NCBI Taxonomy)
                • Description: In Turkmenistan, about 42 to 67% of the sheep and goats were also found to be positive for IgG anti-HEV (Meng et al., 2002).
              • Ovis aries; Ovis ovis; Ovis orientalis aries; Ovis ammon aries; Ovis orientalis :
                • GenBank Taxonomy No.: 9940
                • Scientific Name: Ovis aries; Ovis ovis; Ovis orientalis aries; Ovis ammon aries; Ovis orientalis (NCBI Taxonomy)
                • Common Name: Sheep; domestic sheep; wild sheep; lambs (NCBI Taxonomy)
                • Description: In Turkmenistan, about 42 to 67% of the sheep and goats were also found to be positive for IgG anti-HEV (Meng et al., 2002).
              • Canis familiaris; Canis lupus familiaris; Canis domesticus; Canis canis :
                • GenBank Taxonomy No.: 9615
                • Scientific Name: Canis familiaris; Canis lupus familiaris; Canis domesticus; Canis canis (NCBI Taxonomy)
                • Common Name: Dog; dogs; beagle dogs; beagle dog (NCBI Taxonomy)
                • Description: In Vietnam, where hepatitis E is endemic, anti-HEV has been detected in 44% of chickens, 36% of pigs, 27% of dogs, and 9% of rats (Meng et al., 2002).
              • Macaca mulatta :
                • GenBank Taxonomy No.: 9544
                • Scientific Name: Macaca mulatta (NCBI Taxonomy)
                • Common Name: Rhesus monkey; rhesus macaque; rhesus monkeys; rhesus macaques (NCBI Taxonomy)
                • Description: Naturally acquired anti-HEV was also detected in rhesus macaques (Meng et al., 2002).
              • Rodentia :
                • GenBank Taxonomy No.: 9989
                • Scientific Name: Rodentia (NCBI Taxonomy)
                • Common Name: Rodents (NCBI Taxonomy)
                • Description: Anti-HEV also has been detected in wild-caught rats in the United States and other countries. The prevalence of anti-HEV increased in parallel with the estimated age of the rats (Meng et al., 2002).
              • Cervus nippon nippon :
                • GenBank Taxonomy No.: 9863
                • Scientific Name: Cervus nippon nippon (NCBI Taxonomy)
                • Common Name: Sika deer (NCBI Taxonomy)
                • Description: Tei and co-workers in Japan experienced a series of cases of HEV infection among people who had eaten uncooked deer meat. A left over portion of the deer meat, kept frozen was positive for HEV RNA, whose nucleotide sequence was identical to those from the patients. Patients' family members who ate none or very little of the deer meat remained uninfected, thus providing direct evidence for HEV infection to be a zoonosis (Tei et al., 2003).
              • Equus asinus :
                • GenBank Taxonomy No.: 9793
                • Scientific Name: Equus asinus (NCBI Taxonomy)
                • Common Name: Ass; domestic ass; donkey; African wild ass; Somali wild ass; African ass (NCBI Taxonomy)
                • Description: HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
              • Mule :
                • GenBank Taxonomy No.:
                • Scientific Name: Mule (Panda et al., 2006)
                • Common Name: Mule; mules (Panda et al., 2006)
                • Description: HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
              • Herpestidae :
                • GenBank Taxonomy No.: 9697
                • Scientific Name: Herpestidae (NCBI Taxonomy)
                • Common Name: Mongooses (Panda et al., 2006)
                • Description: HEV RNA has been detected in the feces and anti-HEV antibodies have been detected in sera of domestic swine, cattle, donkeys, deer, rodents, mules, mongoose and other animals (Panda et al., 2006).
      
      
   2. Infection Process:
      
      No infection process information is currently available here.
      
      
   3. Disease Information:
      
      No disease information is currently available here.
      
      
   4. Prevention:
      
      No prevention information is currently available here.
      
      
   5. Model System:
      
      No model system information is currently available here.
      
      
3. Birds:
   1. Taxonomy Information:
      1. Species:
         1. Birds :
            • GenBank Taxonomy No.: 8782
            • Scientific Name: Aves (NCBI Taxonomy)
            • Description: In 2001 a virus associated with a distinct syndrome in chickens in the USA, hepatitis-splenomegaly syndrome (HS), was partially sequenced and shown to be genetically related to hepatitis E viruses (Goens and Perdue, 2004).
            • Variant(s):
              • Gallus gallus :
                • GenBank Taxonomy No.: 9031
                • Scientific Name: Gallus gallus (NCBI Taxonomy)
                • Common Name: Chicken; bantam; chickens; dwarf Leghorn chickens; red junglefowl (NCBI Taxonomy)
                • Description: In an endemic area of Nepal, naturally acquired HEV-specific antibodies were detected in chickens, pigs and rats (Safary, 2001).
      
      
   2. Infection Process:
      
      No infection process information is currently available here.
      
      
   3. Disease Information:
      
      No disease information is currently available here.
      
      
   4. Prevention:
      
      No prevention information is currently available here.
      
      
   5. Model System:
      
      No model system information is currently available here.
      
      

IV. Labwork Information

A. Biosafety Information:

1. General biosafety information :
   • Biosafety Level: Biosafety level 2 (Huang et al., 2005)
   • Applicable: Biosafety level 2 practices and containment for activities with infected materials; Animal Biosafety level 2 for activities using naturally or experimentally infected chimpanzees (MSDS, Public Health Agency of Canada, 2001).
   • Precautions:
     • Laboratory coat; gloves when direct contact with infectious materials is unavoidable; gloves and gown for work in biosafety cabinet. Animal care personnel should wear gloves and take other appropriate precautions to avoid possible fecal-oral exposure; good personal hygiene and thorough washing of hands (MSDS, Public Health Agency of Canada, 2001).
   • Disposal:
     • SPILLS: Allow aerosols to settle, wearing protective clothing, gently cover spill with paper towel and apply 1% sodium hypochlorite, starting at perimeter and working towards the centre; allow sufficient contact time (30 min) before clean up (MSDS, Public Health Agency of Canada, 2001). DISPOSAL: Decontaminate before disposal; steam sterilization, incineration, chemical disinfection (MSDS, Public Health Agency of Canada, 2001). STORAGE: In sealed containers that are appropriately labeled (MSDS, Public Health Agency of Canada, 2001).

B. Culturing Information:

1. Hepatocarcinoma cell line (PLC/PRF/5) :
   1. Description: Tanaka and co-workers, using a fecal suspension with high load of Hepatitis E virus (HEV) (2.0x10(7) copies ml(-1), genotype 3), developed an efficient cell-culture system for HEV in hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4x10(4) copies of HEV were inoculated on monolayers of PLC/PRF/5 cells, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1x10(5) copies per ml on day 60. When 8.6x10(5) copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6x10(7) copies per ml on day 60 (Tanaka et al., 2007).
      
      
   2. Medium:
      1. Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % (v/v) heat-inactivated fetal calf serum (FCS), 100 U penicillin G/ml, 100 ug streptomycin/ml and 2.5 ug amphotericin B/ml, at 37 C in a humidified 5 % carbon dioxide atmosphere. The maintenance medium used for virus culturing consisted of 50 % DMEM and 50 % medium 199 containing 2 % (v/v) heat-inactivated FCS and 30 mM Magnesium chloride at final concentration; other supplements were the same as in the growth medium (Tanaka et al., 2007).
   3. Optimal Temperature: 37 C
   4. Lower Temperature: 35.5 C
   5. Note: For virus infection, confluent cells were trypsinized and diluted 1 : 4 in medium and 2.0 ml was added to wells (diameter of 3.5 cm) of a six-well microplate 1 or 2 days before virus infection (Tanaka et al., 2007).

C. Diagnostic Tests :

1. Organism Detection Tests:
   
   No organism detection tests available here.
   
   
2. Immunoassay Tests:
   1. Enzyme Immunoassay:
      1. Time to Perform: 1-to-2-days
      2. Description: The "golden standard" for diagnosis of HEV infection should be ideally the detection of viremia, by detection of either the HEV genome or the capsid protein. However, detection of HEV RNA by RT-PCR is confined to specialist laboratories and no kit for detection of HEV antigen is available to date. An indirect sandwich EIA was developed for detection of HEV antigen in serum and fecal samples. Approximately 44.6% (33/74), 28.6% (50/175), and none (0/27) of sera positive for anti-HEV IgM alone, both anti-HEV IgG and IgM, and anti-HEV IgG alone, respectively, were positive for HEV antigen using the EIA and there was no cross-reactivity with sera from patients with acute hepatitis A, B, and C. Some HEV antibody positive sera were tested for both HEVRNA and antigen in parallel. The concordance of HEV RNA and antigen was 81.0% (34/42). All the positive PCR products were cloned and sequenced and found to belong to HEV genotype 4 (Zhang et al., 2006).
   2. Enzyme-linked Immunosorbent Assay (ELISA):
      1. Time to Perform: 1-to-2-days
      2. Description: An enzyme-linked immunosorbent assay (ELISA) for detecting anti-HEV antibodies was carried out using a recombinant ORF2 antigen generated on the basis of the gene sequence of genotype IV HEV for an IgG-type anti-HEV antibody (VIRAGENT anti-HEV antibody (human IgG), Cosmic Corporation, Tokyo, Japan) and an IgM-type anti-HEV antibody (VIRAGENT anti-HEV antibody (human IgM), Cosmic Corporation, Tokyo, Japan). A cut-off index of positivity for the IgG-type anti-HEV antibody was set at 13 according to the manufacturer's instructions, and patients with indices of 13 or higher were considered positive for the IgG-type anti-HEV antibody. A cut-off index of positivity for the IgM-type anti-HEV antibody was set at 30 according to the manufacturer's instructions, and patients with indices of 30 or higher were considered positive for the IgM-type anti-HEV antibody (Kikuchi et al., 2006).
   3. Enzyme Linked Immunosorbent Assay (ELISA):
      1. Time to Perform: 1-to-2-days
      2. Description: Acute hepatitis E (AHE) has rarely been reported in industrialized countries, but the rate of seroprevalence of hepatitis E virus (HEV) antibodies (anti-HEV) is inappropriately high. The sensitivity and specificity of the assay used to test for immunoglobulin G (IgG) and IgM anti-HEV have not been well established in areas where hepatitis E is not endemic (hereafter referred to as "nonendemic areas"). Serum samples from 13 AHE patients, 271 healthy subjects, and 160 other liver disease patients in Taiwan were collected and tested for HEV RNA by reverse transcription (RT)-PCR and for IgG and IgM anti-HEV by enzyme-linked immunosorbent assays. The sensitivities of IgG and IgM anti-HEV (relative to RT-PCR) were 86.7 and 53.3%, respectively. The specificities of IgG and IgM anti-HEV assays for diagnosing AHE were 92.1 and 98.6%, respectively. The rate of seroprevalence of IgG anti-HEV was 11% among healthy subjects in this nonendemic area, and it increased with age. In summary, IgG anti-HEV is a good diagnostic test for screening for AHE in nonendemic areas. The high rate of prevalence of anti-HEV in healthy subjects indicates that subclinical infection may exist (Lin et al., 2000).
   4. Solid-Phase Enzyme Immunoassay:
      1. Time to Perform: 1-to-2-days
      2. Description: Serum samples collected from 68 patients (age, mean +/- the standard deviation [SD], 56.3 +/- 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 +/- 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 +/- 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection (Takahashi et al., 2005).
   5. Enzyme-linked Immunosorbent Assay:
      1. Time to Perform: 1-to-2-days
      2. Description: To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immunodominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77-100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1-16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30-40 days after onset of jaundice and decreased to 1:1,778 3-4 months after the onset of jaundice. For patients tested 6-8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1-40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3-4 and 6-12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases (Favorov et al., 1996).
   6. Enzyme-linked Immunosorbent Assay:
      1. Time to Perform: 1-to-2-days
      2. Description: Assay for HEV antibody by enzyme-linked immunosorbent assay (ELISA): Immunoglobulin G (IgG) and IgM antibodies to HEV were measured by ELISA. The ELISA to detect anti-HEV using virus-like particles expressed by a recombinant baculovirus was performed as reported previously by Li et al., 1997 (Abe et al., 2004).
   
   
3. Nucleic Acid Detection Tests: :
   1. RT-Nested PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Total RNAs were extracted from the serum sample with the TRIZOL LS reagent. The RNA was reverse transcribed with antisense primer (HE040; 5'-CCC TTR TCC TGC TGA GCR TTC TC-3' (R = A or G)) specific for the HEV ORF2 sequence. A part of the ORF2 sequence was amplified with nested PCR using the primer pair HE044 (5'-CAA GGH TGG CGY TCK GTT GAG AC-3'[H = A, T, or C; Y = T or C; and K = G or T)) and HE040 in the first round and HE110-2 (mixture of three primers, 5'-GYT CKG TTG AGA CCT CYG GGG T-3', 5'-GYT CKG TTG AGA CCA CGG GYG T-3', and 5'-GYT CKG TTG AGA CCT CTG GTG T-3') and HE041 (5'-TTM ACW GTC RGC TCG CCA TTG GC-3'(M _ = A or C, W = A or T)) in the second round (Kikuchi et al., 2006).
      3. Primers:
         • HE044, HE040
           • Forward: HE044: 5'-CAA GGH TGG CGY TCK GTT GAG AC-3' (H = A, T, or C; Y = T or C; and K = G or T)
           • Reverse: HE040: 5'-CCC TTR TCC TGC TGA GCR TTC TC-3' (R = A or G))
           • Product
             • Size: 506 bp
         • HE110-2 (sense primer; mixture of three sequences), HE041
           • Forward: HE110: 5'-GYT CKG TTG AGA CCT CYG GGG T-3' HE111: 5'-GYT CKG TTG AGA CCA CGG GYG T-3' HE112: 5'-GYT CKG TTG AGA CCT CTG GTG T-3'
           • Reverse: HE041: 5'-TTM ACW GTC RGC TCG CCA TTG GC-3' (M _ = A or C, W = A or T)
           • Product
             • Size: 458 bp
   2. RT-PCR/Nested-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Serum HEV RNA was reverse transcribed to generate cDNA using random primers. The cDNA was divided for PCR using different sets of primers. Nested PCR used to detect HEV RNA with two sets of primers (F1 and R1 in the first round and F2 and R2 in the second round). Another two sets of primers (set 1 primers, external 3,156 and 3,157, internal 3,158 and 3,159; set 2 primers, external 3,160 and 3,161, internal 3,162 and 3,163) were also used in nested PCR. The sensitivity of RT-PCR for detecting HEV RNA is 10 copies. The amplified PCR products had been cloned, sequenced, and deposited in GenBank previously (31, 32) (Lin et al., 2000).
      3. Primers:
         • Nested PCR (First round) F1 and R1
           • Forward: F1: 5'-GCTATTATGGAGGAGTGT-3' (Wu et al., 1998)
           • Reverse: R1: R1 5'-CAGGGCCCCAATTCT-3 (Wu et al., 1998)
         • Nested PCR (Second round) F2 and R2
           • Forward: F2: 5'-GCGTGGATCTTGCAG-3' (Wu et al., 1998)
           • Reverse: R2: 5'-TTCAACTTCAAGCCACAG-3' (Wu et al., 1998)
         • set 1 primers (external): 3,156 and 3,157
           • Forward: 3156: 5'-AAT(C)TATGCC(A)CAGTACCGGGTTG-3' [position 5687-5708] (Meng et al., 1997(a))
           • Reverse: 3157: 5'-CCCTTATCCTGCTGAGCATTCTC-3' [position 6395-6417] (Meng et al., 1997(a))
         • set 1 primers (internal): internal 3158 and 3159
           • Forward: 3158: 5'-GTT(C)ATGC(T)TT(C)TGCATACATGGCT-3' [position 5972-5993] (Meng et al., 1997(a))
           • Reverse: 3159: 5'-AGCCGACGAAATC(T)AATTCTGTC-3' [position 6298-6319] (Meng et al., 1997(a))
         • set 2 primers (external)
           • Forward: 3160: 5'-GCCGAGTAT(C)GACCAGTCCACTTA-3' [position 6578-6600] (Meng et al., 1997(a))
           • Reverse: 3161: 5'-AT(C)AACTCCCGAGTTTTACCCACC-3' [position 7105-7127] (Meng et al., 1997(a))
         • set 2 primers (internal)
           • Forward: 3162: 5'-TGGTT(G)AATGTT(A)GCGACC(T)GGCGCG-3' [position 6645-6667] (Meng et al., 1997(a))
           • Reverse: 3163: 5'-GCTCAGCGACAGTA(T)GACTGG(A)AAA-3' [position 7063-7085] (Meng et al., 1997(a))
   3. Nested RT-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The isolates of Korean human HEV were identified in serum samples by a nested RT-PCR technique. Nested PCR and reverse transcriptase PCR were developed based on the nucleotide sequences of open reading frame 2 (ORF 2) of U.S. and Japanese HEV isolates from humans and Korean HEV isolates from swine (Ahn et al., 2005).
      3. Primers:
         • External set of primers
           • Forward: 5'-TCC CCG CTT ACA TCA TCT GTT GC-3'
           • Reverse: 5'-CTT TAC TGT TGG CTC GCC ATT GG-3'
           • Product
             • Size: 813 bp
         • Nest set of primers
           • Forward: 5'-AAC CCT CTC TTG CCT CTT CAG G-3'
           • Reverse: 5'-AGG GCG GGA GTA AAA CAG TTG-3'
           • Product
             • Size: 720 bp
   4. Nested RT-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A new method for the serological diagnosis of hepatitis E virus (HEV) infection based on neutralization of the virus in cell culture was developed. The test involves a short incubation of the virus in the presence of the serum sample to be tested and permissive cells. With viral replication being limited and without a cytopathic effect, viral growth in cells is evaluated by reverse transcription and PCR. The specificity of the test was established by studying sera from healthy individuals and patients with hepatitis living in France, where autochthonous hepatitis E is unknown. Neutralizing antibodies were found in 79% of patients during an outbreak of hepatitis E and in 43% of patients with sporadic, acute non-A, non-B (without anti-hepatitic C virus antibodies) hepatitis. This neutralization assay is proposed as a confirmatory test for the available enzyme-linked immunosorbent assay (ELISA), which is now recognized as giving many false-positive reactions, and to improve identification of new hepatitis viruses since false-negative reactions with HEV ELISA are also encountered (Meng et al., 1997(b)).
      3. Primers:
         • S1, A1
           • Forward: S1: 5'-GAG GCA GGC ACA ACT AAA GC-3'
           • Reverse: A1: 5'-AAG AAG GGG GGC ACA AG-3'
         • S2, A2 (nested primers)
           • Forward: S2: 5'-GCA CCG GGT CGC TAT TTC-3'
           • Reverse: A2: 5'-TGA AGC TCA GCG ACA GTA GA-3'
           • Product
             • Size: 216 bp
   5. TaqMan RT-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Jothikumar and co-workers developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA in clinical and environmental samples. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1 - 4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120 - 1.2 PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay (Jothikumar et al., 2006).
      3. Primers:
         • JVHEVF; JVHEVR
           • Forward: JVHEVF: 5'-GGTGGTTTCTGGGGTGAC-3'
           • Reverse: JVHEVR: 5'-AGGGGTTGGTTGGATGAA-3'
   6. TaqMan Real time PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Real time PCR based on TaqMan detection was used to identify HEV genome fragments in the serum of patients with positive or questionable anti-HEV serology. HEV RNA was found in 25.4% of cases. The primer pair was selected from the ORF2 region which encodes the virus capsid and a 189-base long product was amplified (Mansuy et al., 2004).
      3. Primers:
         • Pair of primers
           • Forward: Sense primer: 5'GACAGAATTRATTTCGTCGGCTGG3'
           • Reverse: Anti-sense primer: 5'TGYTGGTTRTCATAATCCTG3'
           • Product
             • Size: 189 bp
   7. Taq-Man (RT)-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A single real-time reverse transcription (RT)-PCR assay with Taq- Man technology has been validated which uses only one set of primers and probe within the ORF2 HEV region (nt 5207 - 5292) for the detection and quantification of the four major genotypes of HEV. This assay proved to be as efficient as the conventional RT-PCR methodology for the detection of HEV in clinical samples testing positive previously. The real-time RT-PCR and conventional RT-PCR were performed comparatively on 60 pairs of sera and stools collected during a recent outbreak of hepatitis E in Darfur. The real-time (RT)-PCR assay was 10- to 100-fold sensitive than for conventional RT-PCR assays used in this study with a range quantitation from 1.8 x 10(1) to 7.2 x 10(3) RNA copies/ml in clinical samples (serum and stools) (Enouf et al., 2006).
      3. Primers:
         • TaqHEV-F, TaqHEV-F
           • Forward: TaqHEV-F: 5'-GCCCGGTCAGCCGTCTGG-3' [5207 - 5224] (b)
           • Reverse: TaqHEV-R: 5'-CTGAGAATCAACCCGGTCAC-3' [5273 - 5292] (b)
   8. RT-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The use of microspin columns for extraction of hepatitis E virus (HEV) RNA from stool and serum specimens for reverse transcription-polymerase chain reaction (RT-PCR) and compares this method with the glass powder method. The microspin column method was found to be 1- to 2-log more sensitive in detecting HEV RNA than the glass powder method and had better reproducibility. The microspin column method also detected HEV RNA in a larger number of specimens than the glass powder method from among a panel of serum and stool specimens. Use of this method may allow better assessment of viremia and fecal excretion in patients (Aggarwal and McCaustland, 1998).
      3. Primers:
         • Burma strain
           • Forward: 5'-TGT TTG AGA ATG ACT TTT CTG AGT TTG ACT-3'
           • Reverse: 5'-TAA TAA CGG CCA TAT TCC AGA CAG TAT TCC-3'
           • Product
             • Size: 237 bp
         • Mexico strain
           • Forward: 5'-GAC TCA GTAT TCT CTG CTG CCG TGG-3'
           • Reverse: 5'-CCA TGT TCC ACA CCG TAT TCC AGAG-3'
           • Product
             • Size: 272 bp
   9. Reverse transcription-PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Stools and sera collected from humans with acute HEV infections during epidemic and sporadic cases were analyzed by reverse transcription-PCR. Two methods for RNA purification were compared. Proteinase K digestion and phenolchloroform extraction were more efficient than guanidinium isothiocyanate extraction in improving the sensitivity and specificity for the detection of HEV genomes (Turkoglu et al., 1996).
      3. Primers:
         • Outer primers: ET-F1, ET-R1
           • Forward: ET-F1: 5'-GCT CAT TAT GGA GAG AGT GTG G-3'
           • Reverse: ET-R1: 5'-CAG GGC CCC CAA GTT CTT CT-3'
         • Internal sense and antisense: ET-F2, ET-R2
           • Forward: ET-F2: 5'-GCG TGG ATC TCT GCA GGC C-3'
           • Reverse: ET-R2: 5'-TTC AAC TTC AAG ACC ACA GCC-3'
   
   
4. Other Types of Diagnostic Tests:
   
   No other tests available here.
   
   

V. References

A. Journal References:

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Aggarwal and McCaustland, 1998: Aggarwal R, McCaustland KA. Hepatitis E virus RNA detection in serum and feces specimens with the use of microspin columns. J Virol Methods. 1998; 74(2): 209 - 213. [PubMed: 9779621].

Aggarwal et al., 2001: Aggarwal R, Kamili S, Spelbring J, Krawczynski K. Experimental studies on subclinical hepatitis E virus infection in cynomolgus macaques. J Infect Dis. 2001; 184(11): 1380 - 1385. [PubMed: 11709779].

Ahn et al., 2005: Ahn JM, Kang SG, Lee DY, Shin SJ, Yoo HS. Identification of novel human hepatitis E virus (HEV) isolates and determination of the seroprevalence of HEV in Korea. J Clin Microbiol. 2005; 43(7): 3042 - 3048. [PubMed: 16000413].

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author et al., 2007: Michitaka K, Takahashi K, Furukawa S, Inoue G, Hiasa Y, Horiike N, Onji M, Abe N, Mishiro S. Prevalence of hepatitis E virus among wild boar in the Ehime area of western Japan. Hepatol Res. 2007; 37(3): 214 - 220. [PubMed: 17362304].

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Bradley et al., 1987: Bradley DW, Krawczynski K, Cook EH Jr, McCaustland KA, Humphrey CD, Spelbring JE, Myint H, Maynard JE. Enterically transmitted non-A, non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease-associated 27- to 34-nm virus-like particles. Proc Natl Acad Sci U S A. 1987; 84(17): 6277 - 6281. [PubMed: 3114746].

Buisson et al., 2000: Buisson Y, Grandadam M, Nicand E, Cheval P, van Cuyck-Gandre H, Innis B, Rehel P, Coursaget P, Teyssou R, Tsarev S. Identification of a novel hepatitis E virus in Nigeria. J Gen Virol. 2000; 81(Pt 4): 903 - 909. [PubMed: 10725415].

CDC, 1999: CDC. , Prevention of hepatitis A through active or passive immunization: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 1999; 48(RR12): 1 - 37. [PubMed: 10543657].

Chauhan et al., 1994: Chauhan A, Dilawari JB, Kaur U, Ganguly NK, Bushnurmath S, Chawla YK. Atypical strain of hepatitis E virus (HEV) from north India. J Med Virol. 1994; 44(1): 22 - 29. [PubMed: 7798881].

Clayson et al., 1998: Clayson ET, Vaughn DW, Innis BL, Shrestha MP, Pandey R, Malla DB. Association of hepatitis E virus with an outbreak of hepatitis at a military training camp in Nepal. J Med Virol. 1998; 54(3): 178 - 182. [PubMed: 9515765].

Corwin et al., 1996: Corwin AL, Khiem HB, Clayson ET, Pham KS, Vo TT, Vu TY, Cao TT, Vaughn D, Merven J, Richie TL, Putri MP, He J, Graham R, Wignall FS, Hyams KC. A waterborne outbreak of hepatitis E virus transmission in southwestern Vietnam. Am J Trop Med Hyg. 1996; 54(6): 559 - 562. [PubMed: 8686771].

Emerson et al., 2002: Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH. Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol. 2006; 87(Pt 3): 697 - 704. [PubMed: 16476993].

Enouf et al., 2006: Enouf V, Dos Reis G, Guthmann JP, Guerin PJ, Caron M, Marechal V, Nicand E. Validation of single real-time TaqMan PCR assay for the detection and quantitation of four major genotypes of hepatitis E virus in clinical specimens. J Med Virol. 2006; 78(8): 1076 - 1082. [PubMed: 16789018].

Favorov et al., 1996: Favorov MO, Khudyakov YE, Mast EE, Yashina TL, Shapiro CN, Khudyakova NS, Jue DL, Onischenko GG, Margolis HS, Fields HA. IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein. J Med Virol. 1996; 50(1): 50 - 58. [PubMed: 8890041].

Ghabrah et al., 1998: Ghabrah TM, Tsarev S, Yarbough PO, Emerson SU, Strickland GT, Purcell RH. Comparison of tests for antibody to hepatitis E virus. J Med Virol. 1998; 55(2): 134 - 137. [PubMed: 9598934].

Goens and Perdue, 2004: Goens SD, Perdue ML Hepatitis E viruses in humans and animals. Anim Health Res Rev. 2004; 5(2): 145 - 156. [PubMed: 15984321].

Guthmann et al., 2006: Guthmann JP, Klovstad H, Boccia D, Hamid N, Pinoges L, Nizou JY, Tatay M, Diaz F, Moren A, Grais RF, Ciglenecki I, Nicand E, Guerin PJ. A large outbreak of hepatitis E among a displaced population in Darfur, Sudan, 2004: the role of water treatment methods. Clin Infect Dis. 2006; 42(12): 1685 - 1691. [PubMed: 16705572].

He et al., 2002: He J, Innis BL, Shrestha MP, Clayson ET, Scott RM, Linthicum KJ, Musser GG, Gigliotti SC, Binn LN, Kuschner RA, Vaughn DW. Evidence that rodents are a reservoir of hepatitis E virus for humans in Nepal. J Clin Microbiol. 2002; 40(12): 4493 - 4498. [PubMed: 12454141].

Hsieh et al., 1999: Hsieh SY, Meng XJ, Wu YH, Liu ST, Tam AW, Lin DY, Liaw YF. Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. J Clin Microbiol. 1999; 37(12): 3828 - 3834. [PubMed: 10565892].

Huang et al., 1992: Huang CC, Nguyen D, Fernandez J, Yun KY, Fry KE, Bradley DW, Tam AW, Reyes GR. Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology. 1992; 191(2): 550 - 558. [PubMed: 1448913].

Huang et al., 2002: Huang FF, Haqshenas G, Shivaprasad HL, Guenette DK, Woolcock PR, Larsen CT, Pierson FW, Elvinger F, Toth TE, Meng XJ. Heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the United States. J Clin Microbiol. 2002; 40(11): 4197 - 4202. [PubMed: 12409397].

Huang et al., 2005: Huang YW, Haqshenas G, Kasorndorkbua C, Halbur PG, Emerson SU, Meng XJ. Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs. J Virol. 2005; 79(3): 1552 - 1558. [PubMed: 15650181].

Hussaini et al., 1997: Hussaini SH, Skidmore SJ, Richardson P, Sherratt LM, Cooper BT, O'Grady JG. Severe hepatitis E infection during pregnancy. J Viral Hepat. 1997; 4(1): 51 - 54. [PubMed: 9031065].

Iqbal et al., 1989: Iqbal M, Ahmed A, Qamar A, Dixon K, Duncan JF, Islam NU, Rauf A, Bryan JP, Malik IA, Legters LJ. An outbreak of enterically transmitted non-A, non-B hepatitis in Pakistan. Am J Trop Med Hyg. 1989; 40(4): 438 - 443. [PubMed: 2496611].

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B. Book References:

Purcell and Emerson, 2002: Purcell Robert H, Emerson Suzanne U. Hepatitis E Virus. 799 - 840. In: Knipe David M., Howley Peter M. Fields Virology 2001. Lippincott Williams & Wilkins, Philadelphia . Baltimore . New York . London . Buenos Aires . Hong Kong . Sydney . Tokyo.

C. Website References:

CDC2006a: Hepatitis E virus [ http://www.cdc.gov/ncidod/diseases/hepatitis/slideset/hep_e/slide_1.htm ].

CDC2006b: Hepatitis E Virus Infection [ http://www.cdc.gov/ncidod/diseases/hepatitis/slideset/hep_e/slide_3.htm ].

MSDS, Public Health Agency of Canada, 2001: Material Safety Data Sheet - Infectious Substances: Hepatitis A virus [ http://www.phac-aspc.gc.ca/msds-ftss/msds75e.html ].

NCBI Taxonomy: Hepatitis E virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=12461&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Big liver and spleen disease virus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=301242&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus (isolate rhesus) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=31766&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus (strain Burma) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=31767&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus (strain Mexico) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=31768&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus (strain Myanmar) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=31769&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus (strain Pakistan) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=33774&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus type 1 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=185579&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Hepatitis E virus type 4 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=185580&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Genome: Hepatitis E virus, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=10157 ].

NCBI Taxonomy: Sus scrofa [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9823&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Bos taurus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9913 ].

NCBI Taxonomy: Capra hircus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9925 ].

NCBI Taxonomy: Rodentia [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9989&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Cervus nippon [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9863&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Entrez: Hepatitis E virus, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=10157 ].

NCBI Taxonomy: Gallus gallus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Equus asinus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9793&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Homo sapiens [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Ovis aries [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9940&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Canis familiaris [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9615&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Macaca mulatta [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9544 ].

NCBI Taxonomy: Metazoa [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=33208&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Aves [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8782&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

NCBI Taxonomy: Geographic distribution of Hepatitis E [ http://www.cdc.gov/ncidod/diseases/hepatitis/slideset/hep_e/slide_5.htm ].

NCBI Taxonomy: Herpestidae [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9697&lvl=3&lin=f&keep=1&srchmode=1&unlock ].

D. Thesis References:

Huang, 2004: Huang Fang-Fang; . Molecular Characterization of Animal Strains of Hepatitis E Virus (HEV): Avian HEV and Swine HEV. 2004. Doctor of Philosophy in Veterinary Medical Sciences. Virginia Polytechnic Institute and State University. Blacksburg, VA (USA).

Sun, 2005: Sun Zhifeng; . Cross-Species Infection and Characterization of Avian Hepatitis E Virus. 2005. Doctor of Philosophy in Veterinary Medical Sciences. Virginia Polytechnic Institute and State University. Blacksburg, VA (USA).

VI. Curation Information

• Curators: Herman Formadi
• Date: 05-07-2007
• Version: 1.0
• Note: PIML_05-07-2007
• Contact information:
  • Email: pathinfo@vbi.vt.edu